1. Field of the Invention
The present invention relates to a method for measuring the copy number of a chromosome, gene or specific nucleotide sequence, comprising the steps of: (a) mixing a homozygous DNA with a test sample DNA; (b) analyzing the DNA mixture by means of SNP array; and (c) determining the copy number of a chromosome, gene, or specific nucleotide sequence by measuring the difference in signal output from the homozygous DNA and the test sample DNA.
2. Description of the Related Art
Changes in specific chromosomal sequences are frequently implicated in human diseases and syndromes. Such changes include the addition or the deletion of one entire chromosome as in Down's syndrome, deletions of several million base pairs as in DiGeorge syndrome, and deletions or duplications of small chromosomal fragments as in Becker or Duchenne muscular dystrophy. A subtelomeric deletion is also frequently reported in mental retardation patients (Lamb et al., 1989). In addition, chromosomal regions of specific genes such as BRCA1 or MLH1/MLH2 are commonly changed in tumors, which is known to be important for gene expressions (Petrij-Bosch et al., 1997; Wijnen et al., 1998). An analysis of the copy number change of genes can be important for the treatment of cancer patients, as can be seen from the example of using ERBB2-specific antibodies to treat a breast cancer patient having ERBB2 gene amplified (Leyland-Jones and Smith, 2001).
At present, many techniques are used to determine the copy number of chromosomal changes. The most standardized method of measuring the number and structural changes of chromosomes is a karyotyping method. According to this method, it is required to culture the patient's blood, fibroblast or amniotic cells, and much time and manpower are necessary to interpret the result thereof. The karyotyping method usually can detect 1 mega base or more of the chromosomal changes only. This sensitivity issue can be made up for with a fluorescent in situ hybridization (FISH) method. However, the FISH method also requires much time and manpower and does not usually measure the changes of four or more different target genes at a time (Klinger et al., 1992). In addition, a multicolor chromosome painting method is introduced as a method for automatization of the karyotyping. The method allows the deletion, duplication or translocation of the chromosome to be easily detected by labeling portions of each chromosome with fluorescent materials of different colors (U.S. Pat. No. 6,066,459). Although the multicolor chromosome painting method increases the sensitivity somewhat, compared to the karyotyping method, it basically needs a cell culture and a post-process which are required for the karyotyping.
In order to overcome the requirements of time and manpower, several molecular methods have been recently developed to detect the chromosomal changes. Array based-comparative genomic hybridization (CGH) is one of the most promising methods and many trials have been attempted for application to the diagnosis on genetic diseases or the detection of chromosomal changes in cancer tissues (Pinkel et al, 1998; U.S. Pat. Nos. 6,197,501 and 6,159,685). This method immobilizes BAC clones on a substrate surface to form an array, and pre-labeled standard DNA and sample DNA are hybridized to the array. According to the method, a relative amount of signals from the standard and sample DNAs is compared to detect the chromosomal changes such as deletion or duplication.
In addition, there is a method of determining the copy number by measuring the relative amplification with multiplex PCR method (Rahil et al., 2002). As a modified method thereof, a multiplex ligation-dependent probe amplification (MLPA) was recently introduced (Schouten et al., 2002; Patent number WO9615271).
Loss of heterozygosity (LOH) is the most common method to detect deletion or duplication of a chromosome. For research of LOH, microsatellite markers (Call et al., 1990) have been used. However, the LOH method using microsatellite markers cannot distinguish whether a chromosomal change is deletion or duplication, except for in the case of homozygous deletion.
Pont-Kindon and Lyon (2003) reported another method of using SNP to detect a chromosomal abnormality. They used a melting curve analysis to detect the relative amount of heterozygous alleles. The method detects that there is a trisomy when the relative amount of two alleles is different from a normal ratio.
In addition, there was introduced a method of detecting deletion or duplication of chromosome by means of SNP array (Lindblad-Toh et al., 2000). The method has enormous potential that the array can be extended to detect a large number of SNPs. However, the SNP array for the detection of chromosomal changes has the limitation that it requires the presence of heterozygous alleles in the test DNA. Common SNPs are those that are present in more than 5% of the population, but the number of SNPs and heterozygote frequency used for CNV (copy number variation) are low in a single gene. Therefore, there is an urgent need for improvement of the method in order to detect specific SNPs of interest.
Therefore, the present inventors have made an effort to solve the above problems and to develop more effective methods for measuring the copy number of a chromosome, gene or specific nucleotide sequence. Finally, the present inventors developed a method comprising the steps of mixing a homozygous DNA with a test sample DNA and analyzing the DNA mixture by means of SNP array capable of measuring rare SNP to measure the difference in signal output from each sample. They found that the method is able to allow more accurate values to be obtained compared to the other molecular methods of determining the copy number of a specific gene as well as remarkably reduce costs and required manpower, thereby completing the present invention.